Hzl (H-2bml) is a strain derived from C57BL/6 (H-2b) carrying a mutation in the H-2K gene. When Hzl mice are infected with vaccinia virus, cytotoxic cells are generated that kill virus infected syngeneic target cells but not target cells carrying H-2Kb. In contrast, when spleen cells from Hzl mice are sensitized in vitro with trinitrophenyl (TNP)-modified spleen cells, cytotoxic cells are generated that kill TNP-target cells carrying the H-2Kb gene. We have shown that the Hzl anti-TNP effector cells lyse H-2Kb target cells that are first infected with vaccinia virus and then modified with TNP, demonstrating that virus does not mask cross reactive determinants shared by TNP-H-2Kb and TNP-H-2Kbml. The specificity in H-2 unrestricted cytotoxic T cells was analysed using mice that differ only at the Qa-l/Tla interval. Our results demonstrate that there are at least two genes controlling target antigens; one in the Qa-l/Tla region, the other not. In certain strain combinations, we showed that factors in addition to Qa-l/tla may be required to generate anti-Qa-l/Tla effector cell activity. B10.A animals, rendered tolerant to B10.M (H.2f) spleen cells, when challenged with A.CA (H-2f) spleen cells both in vivo and in vitro, generate cytotoxic effector cells that have specificity for the A strain minor histocompatibility antigens in the context of H-2f haplotype. These data indicate that at least two sets of T cells coexist in tolerant animals, one capable of recognizing antigens in the context of the host H-2 haplotype, and the other able to recognize antigens in the context of the tolerated H-2 allogeneic haplotype.